Phosphatidylinositol phosphates (PIPs) are membrane-confined secondary messengers that regulate a diverse range of cellular processes (such as membrane trafficking and cytoskeletal dynamics) and bioelectrical signaling through their action on ion pumps and channels. Misregulation of PIPs and PIP binding proteins is implicated in several diseases including various cancers, developmental diseases such as Lowe syndrome and Charcot- Marie-Tooth disease, and Alzheimer's disease. Voltage-sensor-containing phosphatases (VSPs) are voltage- controlled enzymes that remove phosphate groups from PIPs. VSPs are modular proteins comprised of two domains; a membrane-spanning voltage-sensing domain (VSD) and a cytosolic phosphatase domain (PD). The two domains are held together by a 17 amino acid, positively charged intracellular linker that communicates membrane depolarization to the catalytic PD, activating and modulating phosphatase activity. The goal of this project is to understand the mechanism by which transmembrane voltage sensed by the VSD is communicated to the PD using two different hybrid optical-electrophysiological techniques that enable motions of specific protein segments to be monitored in real time: two-electrode voltage-clamp fluorometry (VCF) in intact cells and patch-clamp fluorometry (PCF) on excised patches. Novel variations on these techniques are being developed for this project that will make it possible, for the first time, to measure the motions of intracellular domains, so that VSD-PD coupling and PD gating can be monitored directly. Insight into the mechanism by which transmembrane voltage is communicated to enzymatic activity in VSPs will open up the possibility of pharmacological intervention to adjust their activity level and substrate specificity. Targeting VSPs may be an optimal way to control PIP signaling and address human diseases affected by misregulation of PIPs and PIP binding proteins. Additionally, it is likely that other membrane proteins involved in signaling cascades share similar mechanisms for communicating extracellular stimuli to enzymatic activity. The techniques presented in this proposal, specifically the use of intracellular labels in VCF and PCF, should be applicable to the study of other membrane proteins involved in intracellular signaling.